1. New metal complexes supported by scorpionate and macrocyclic ligands: chemistry and biological studies
Dr.ssa Grazia Papini
School of Advanced Studies
Doctorate course in Chemical Sciences
Cycle XX
Scientific-Sector CHIM/03
Tutor
Prof. Giancarlo Gioia Lobbia 1

2. Well-known Scorpionate Ligands
Bis(pyrazolyl)borates
Tris(pyrazolyl)borates
Tetrakis(pyrazolyl)borates

3. “Nitrogen heterocycles other than pyrazole can be used, such as imidazole, triazole, benzotriazole, thioimidazole, ecc.”
μ3-N,N’,N”
μ2-N,N’
μ4-N4
Tris(imidazolyl)borates
Bis(imidazolyl)borates
Tetrakis(imidazolyl)borates
3-S,S’,B-H
2-S,S’
Poly(triazolyl)borates
Poly(benzotriazolyl)borates
Bis(3-R-2-thioxo-imidazolyl)borates 3

4. Scorpionate ligands with EWG substituents
“Other modifications include changing the substituents on the heterocyclic ring.”
[H2B(pzCOOEt,Me)2]-
[H3B(pzCF3)]-
S. Alidori, M. Pellei, C. Pettinari, C. Santini, B. W. Skelton, A. H. White, Inorg. Chem. Commun., (2004). G. Bandoli, A. Dolmella, G. Gioia Lobbia, G. Papini, M. Pellei, C. Santini Inorg. Chim. Acta (2006)
H. V. R. Dias, S. Alidori, G. Gioia Lobbia, G. Papini, M. Pellei, C. Santini Inorg. Chem. (2007)
Scorpionate ligands with EWG substituents
[H2B(tzNO2)2]-
[H2B(pzNO2)2]-
[H2B(pzCF3)2]-
M. Pellei, F. Benetollo, G.Gioia Lobbia,
S. Alidori, and C. Santini, Inorg. Chem., (2005)
M. Pellei, S. Alidori, G. Papini, G. Gioia Lobbia, J. D. Gorden, H. V. Rasika Dias, C. Santini, Dalton Trans. (2007)

5. “In addition, tripodal ligands can have central atoms other than boron, such as carbon, phosphorus, or silicon….”
RC(pzx)3
RSi(pzx)3
(pzx)3PO
“…..and bearing a coordinating moiety (R') such as acetate, dithioacetate, sulfonate, ethoxide, ”
bdmpza
bdmpzta
bdmpzs 5

6. Rhenium complexes
Versatile chemistry: several oxidation states accessible (from -I to VII); different coordination numbers (from 4 to 8); various donor set available
The similarity between technetium and rhenium chemistry, determined a widespread use of the latter as a technetium surrogate to perform macroscopic chemistry of potential radiopharmaceuticals. In this way, a ‘‘cold’’material (the natural isotopic mixture of 185Re and 187Re) can be advantageously manipulated instead of the radioactive nuclide 99gTc (t1/2 = 2.12 · 105 y, Eβ = 292 keV).
Rhenium has two β- emitters isotopes 186Re (β-max = 1.07 MeV; t1/2 = 90 h) and 188Re (β-max = 2.10 MeV; t1/2 = 17 h) which are of great interest to nuclear medicine due to their physical and nuclear properties finalized to a potential application in the radiopharmaceutical

7. The “metal - fragment” strategy
Bioactive
molecule
Linker
Labile groups M
Stable building -block

8. Re(V) complexes
Metal fragment 8

9. Metal Fragments
ROH(Et3N)
ROH (Et3N) N R e O C l N R e O C l S 2
M. Porchia, G. Papini, C. Santini, G. Gioia Lobbia, M. Pellei, F. Tisato, G. Bandoli, A. Dolmella, Inorg. Chem. 44 (2005) 4045

10. Mixed coordination sphere complexes
E= CO,SO2
n= 2, 3
Structure of the complex
[Re(O)(bdmpza)(OCH2CH2O)]
Structure of the complex [Re(O)(bdmpza)(OCH2CH2CH2O)]
M. Porchia, G. Papini, C. Santini, G. Gioia Lobbia, M. Pellei, F. Tisato, G. Bandoli, A. Dolmella, Inorg. Chem. 44 (2005) 4045

11. Structure of the complex [Re(O)(bdmpza)(mal)]
Marina Porchia,Grazia Papini, Carlo Santini, Giancarlo Gioia Lobbia, Maura Pellei, Francesco Tisato, Giuliano Bandoli, Alessandro Dolmella Inorganica Chimica Acta 359 (2006) 2501–2508.

12. Potential Nitridorhenium complexes

13. Nitridorhenium precursors13 13

14. Pre-carbene ligands
G. Papini, C. Santini, G. Gioia Lobbia, M. Pellei, G. Bandoli, A. Dolmella J. Organomet. Chem. (2008) submitted

15. Liu J. , Chen J. , Zhao J. , Zhao Y. , Li L. , Zhang H
Liu J., Chen J., Zhao J., Zhao Y., Li L., Zhang H., Synthesis 17 (2003) 2661–2666.

17. MIXTURE OF UNCHARACTERIZABLE
[NBu4][ReNCl4]
MIXTURE OF UNCHARACTERIZABLE
PRODUCTS

18. Silver(I) carbene complexes
G. Papini, C. Santini, G. Gioia Lobbia, M. Pellei, G. Bandoli, A. Dolmella J. Organomet. Chem. (2008) submitted 18

19. Carbene transfer reactions
G. Papini, C. Santini, G. Gioia Lobbia, M. Pellei, G. Bandoli, A. Dolmella J. Organomet. Chem. (2008) submitted 19

20. Rhenium derivatives [Ru(p-cymene)Cl2]2 NBu4ReNCl4 CH2Cl2 NBu4ReNCl420
[Ru(p-cymene)Cl2]2

21. [Ru(p-cymene)Cl2]2 [Ru(p-cymene)Cl2]2 Copper and Ruthenium derivatives
Cu(SMe2)Br
CH3CN
[Ru(p-cymene)Cl2]2
CH2Cl2
[Ru(p-cymene)Cl2]2
CH2Cl2
Cu(SMe2)Br
CH3CN 21
[Ru(p-cymene)Cl2]2
[Ru(p-cymene)Cl2]2

22. Fichna et al, Bioconjugate Chem., 14 (2003) 3-17
Copper derivatives
It is an essential trace metal for living organisms
Copper complexes’ activity is extremely wide
Copper has a well-documented coordination chemistry
Several radioactive copper isotopes are available nowadays for biomedical purposes both for radioimaging and targeted radiotherapy
isotope
half-life
imaging
(emission, energy, abundance)
therapy
(emission, energy, range in tissue)
application
Cu-60
20 min
PET
(b+, 873 keV, 93%)
Radiolabelling small molecules
Cu-61
3.3 h
(b+, 527 keV, 62%)
Cu-62
9.7 min
(b+, 1315 keV, 98%)
Cu-64
12.7 h
(b+, 278 keV, 19%)
Radiolabelling small molecules, peptides and antibodies
Cu-66
5.4 min
(b-, 190 keV; 0.95 mm)
for therapy
Cu-67
62.0 h
SPECT
(g, 185 keV, 48%)
Fichna et al, Bioconjugate Chem., 14 (2003) 3-17 22

23. Copper(I) derivatives
C. Marzano, M. Pellei, D. Colavito, S. Alidori, G. Gioia Lobbia, V. Gandin, F. Tisato, and C. Santini, J. Med. Chem., 49 (2006) 7317
P(CH2OH)3
Cells line of ovarian carcinoma (2008) and cis-platino resistent carcinoma cells (C13) 23

24. 31P-NMR = + 9.67 (dbr), - 144.05 (septet)
“CuP4” tipe species
[Cu(CH3CN)4][PF6] + 4 thp  [Cu(thp)4][PF6]
[Cu(CH3CN)4][PF6] + 2 bhpe  [Cu(bhpe)2][PF6]
[Cu(thp)4][PF6]
[Cu(bhpe)2][PF6]
31P-NMR = (dbr), (septet)
[Cu(bhpe)2]+ m/z = 492 (100)
31P-NMR = (q), (septet)
[Cu(thp)4]+ m/z = 560 (6)
[Cu(thp)3]+ m/z = 436 (65)
[Cu(thp)2]+ m/z =312 (100)
C. Marzano, V. Gandin, M. Pellei, D. Colavito, G. Papini, G. Gioia Lobbia, M. Porchia, F. Tisato and C. Santini, J. Med. Chem. 51 (2008) 24

25. bhpe
C. Marzano, V. Gandin, M. Pellei, D. Colavito, G. Papini, G. Gioia Lobbia, M. Porchia, F. Tisato and C. Santini,
J. Med. Chem. 1 (2008) 25

26. Citotoxic activities
Compound
IC50 (µM) ± S.D.
HL60
A549
MCF-7
Daudi
HepG2
A375
CaCo2
HCT-15
HeLa
[Cu(thp)4][PF6]
0.60±0.02
9.11±2.71
11.08±0.52
6.94±0.18
1.26±0.10
4.58±2.41
1.08±0.12
2.00±0.03
8.21±1.50
[Cu(bhpe)2][PF6]
47.40±2.92
57.60±2.19
49.71±2.03
65.5±1.22
78.23±1.11
68.21±1.23
52.50±0.81
57.36±1.31
62.41±1.33
thp
68.63±2.44
72.91±2.44
64.23±4.29
>100
98.71±3.63
88.70±3.88
bhpe
83.72±3.23
71.71±1.64
73.21±1.22
84.11±2.22
91.71±4.01
KPF6
Cisplatin
15.91±1.51
29.21±1.92
19.04±1.51
23.97±2.51
21.50±1.41
20.33±1.33
35.42±1.40
25.34±1.31
10.50±1.51
A549 = lung cancer
CaCo2, HCT-15 = colon cancer
Hela = cervix cancer
MCF-7 = breast cancer
HL60 = leukemia
Daudi = lymphoma
HepG2 = epatoma
A375 = melanoma
IC50 values represent the drug concentrations that reduced the mean absorbance at 570 nm to 50% of those in the untreated control wells. 26

27. Human cervix squamous carcinoma cells Human colon adenocarcinoma cells
Human ovarian adenocarcinoma cells
Cytotoxic activity of [Cu(thp)4][PF6] onto three additional cell line pairs, two of which (2008/C13* ovarian cancer cells and A431/A431-Pt cervix carcinoma cells) selected for their resistance to cisplatin and one (LoVo/LoVoMDR) for its resistance to doxorubicin.
Cross-resistance profiles were evaluated by means of the resistance factor (RF), which is defined as the ratio between IC50 values calculated for the resistant cells and those arising from the sensitive ones.
Compound
2008
IC50 [µM]
C13
R.F.
[Cu(thp)4][PF6]
1.48±0.21
2.88±1.07
1.9
Cisplatin
12.69±1.72
89.18±4.50
7.02
Human cervix squamous carcinoma cells
Compound
A431
IC50 [µM]
A431/Pt
R.F.
[Cu(thp)4][PF6]
14.37±1.41
13.26±0.80
0.92
Cisplatin
22.06±1.62
57.76±1.81
2.61
Human colon adenocarcinoma cells
Compound
LoVo
IC50 [µM]
LoVo-MDR
R.F.
[Cu(thp)4][PF6]
1.54±0.03
2.9±0.1
1.88
Doxorubicin
1.46±2.30
44.89±0.90
30.74

28. Comparison of IC50 values detected by MTT, NR and TB test after incubation of 2008 cells with [Cu(thp)4][PF6] for different exposure times
TB test reveals damage to cell membrane
MTT test mainly reflects damage to mitochondria
The NR assay indicates damage to lysosomes and Golgi apparatus
Lysosomes/Golgi apparatus are more sensitive to complex treatment. On the contrary, the scarce permeability to vital dye indicates that plasma membrane function is still maintained until the late phase of cell death. Lysosomal damage represents the early cellular event associated with copper(I) complex cytototoxicity.

29. 3 h
12 h
24 h
48 h
Cell cycle phases
G1 = GAP 1
S = Synthesis (DNA replication)
G2 = GAP 2
M = mitosis (nuclear and cytoplasmic division)
I = Interphase
untreated cells
cells treated with IC50 of [Cu(thp)4][PF6]
24h
48h
Ctr
Complex 3
p-Value
Apoptosis %
4.24±0.71
2.43±0.66
<0.001
1.21±0.73
15.39±0.96
G1 %
70.92±1.82
60.9±1.35
65.57±1.21
41.03±1.39
G2/M%
20.29±1.11
33.5±1.28
32.62±1.46
37.03±1.12
Percentage of cells in different cell cycle phases as function of time exposure of [Cu(thp)4][PF6], vs control untreated cells

30. Forward scattering (index of cell size) vs side scattering (index of cell granularity) as a function of time in 2008 cells
Mitochondrial energization of treated tumor cells as the retention of a mitochondrial selective cationic fluorescent probe, tetramethyl rhodamine methyl ester (TMRM).
Flow cytometric profiles of 2008 cells untreated (panel A) and treated with (panel B) or 6.25 (panel C) µM of copper(I) complex for 24 h and stained with TMRM (10 nM).
Copper(I) complex induced a massive increase of the TMRM fluorescence reflecting a dramatic alteration of mitochondrial membrane potential that might be correlated with the induction of a G2/M phase cell cycle arrest.
untreated cells
cells treated with IC50 of [Cu(thp)4][PF6]
The coordination of mono-phosphine ligands to copper(I) gives rise to a metallodrug able to inhibit the growth of tumor cells via cell G2/M cell cycle arrest and paraptosis accompanied with the loss of mitochondrial transmembrane potential.

31. Potential Cu(I) radiopharmaceuticals
TPA
64Cu(II)Cl2
Sodium acetate
buffer
Sodium acetate
buffer
THP
(2)
In vitro cell experiments
(1)
Sodium acetate
buffer
Ligand
Cell uptake behavior of complexes 1-4 into EMT-6 mammary carcinoma cells. Error bars not seen are within symbols.
(3)
(4) 31

32. Biodistribution Studies
Biodistribution was carried out on g female BALB/c mice implanted with EMT-6 cells subcutaneously into the left flank. Tumors were allowed to grow for 14 days (approx 0.3 – 0.7 cm3), at which time the animals received 0.20 MBq (~5 μCi) of complex 1 in 100 μL of saline via lateral tail vein injection. Mice were examined at 3 time points (n = 4 per group at 1, 4 and 24 hours). 
S. Alidori, G. Gioia Lobbia, G. Papini, M. Pellei, M. Porchia, F. Refosco, F. Tisato, J.S. Lewis, C. Santini
Journal of Biological Inorganic Chemistry, 13 (2008)
The uptake and retention of activity was high in many non-target tissues lung and liver
Poor blood clearance suggestes breakdown of the complex and binding of 64Cu to serum proteins in vivo.
The heart uptake was high at all time points and there was no clearance from the myocardium over 24 h post-injection potentially due to the
monocationic nature of the complex
Tumor uptake of complex 1 was highest at 1 h and decreased slowly over 24 h. In the same EMT-6 tumor model, uptake of 64Cu-ATSM and 64Cu-PTSM (both of which are clinically tested agents) into the tumor at 40 min post-injection showed lower uptake than that of 1
Tumor uptake of complex 1 is significantly higher than that for [64Cu((EtOCH2CH2)2PCH2CH2P(CH2CH2EtO)2)]+ 32

33. Small animal PET Imaging
Selected axial and coronal images obtained using co-registration techniques demonstrating the uptake of 1 at 1, 2 and 24 h post injection in a mouse with an EMT-6 tumor (arrow) implanted on the flank.The EMT-6 tumors can be easily visualized at all time points
Standard uptake values (SUVs) of 1 in selected organs in EMT-6 tumor bearing mice over 24 h (n = 4).
The uptake in the EMT-6 tumor at 1 h which remained static over 24 h

34. New N-, P- donor ligands 34

35. LiAlH4
1. n-BuLi
2. RX

36. New macrociclic ligands
G. Papini, S. Alidori, J. S. Lewis, D. E. Reichert M. Pellei,, G. Gioia Lobbia, G. B. Biddlecombe, C. J. Anderson,
C. Santini J. Med. Chem. (2008) submitted
P. Blondeau, C. Berse, D. Gravel, Can. J. Chem. 45 (1967) 49. 36

37. Copper(II) complexes
G. Papini, S. Alidori, J. S. Lewis, D. E. Reichert, M. Pellei, G. Gioia Lobbia, G. B. Biddlecombe, C. J. Anderson, C. Santini J. Med. Chem. (2008) submitted 37

38. 64Cu complexes Biodistribution data
The retention of activity in tissues is similar to that observed with 64Cu-cyclam and 64Cu-monooxo-tetrazamacrocyclic complexes, but, on comparison with
64Cu-TETA and 64Cu-DOTA, the uptake
and retention of and are orders-of-magnitude higher.
The poor clearance suggests that the complexes are rapidly degraded in blood and tissues and the 64Cu is sequestered by proteins, and remaining trapped in these tissues hindering clearance. 38

39. Perspectives M
BFCA

40. Conclusions
The monooxo Re(V) core is conveniently stabilized by tripodal scorpionate ligands comprising carboxylate or sulfonate tails, giving a series of intermediate Re(O)(NNO)Cl(X) (X = Cl, OR). To these entities various bidentate ligands (BID) can be attached to produce "3 + 2" mixed ligand compounds.
Hydrophilic ‘cold’ Cu(I)-complexes have shown significant antiproliferative activity in vitro on a series of tumor cell lines, also resistance to cisplatin, showing a different pathway of action from that of cisplatin.
Hydrophylic ‘hot’ 64Cu(I) monophosphine complexes were evaluated as a basis for a new class of copper radiopharmaceuticals. [64Cu(thp)4]+ = building-block for new radiopharmaceuticals, perhaps the first time such a method has been used in the production of Cu-radiopharmaceuticals.
Novel macrocyclic ligands, based on the L,L-ethylenedicysteine skeleton, have been prepared in view of the attractive opportunity to use them as bifunctional chelators for copper nuclides. This is the first report of 64Cu labeled to this form (N2S2) macrocyclics. Although the in vivo biodistribution of complexes suggests dissociation of the 64Cu from the chelates, these new ligands platform offers the potential as a basis for further development to improve the in vivo stability.

41. Prof. Alessandro Dolmella Dr.ssa Cristina Marzano
Partners and Acknowledgements
Prof. Giancarlo Gioia Lobbia
Prof. Carlo Santini
Dr.ssa Maura Pellei
Dr. Simone Alidori
Prof. Jason S. Lewis
Carolyn J. Anderson
Dr. Franco Benetollo
ICIS-CNR, Padova
Prof. Giuliano Bandoli
Prof. Alessandro Dolmella
Dr.ssa Cristina Marzano
Dip. di Scienze Farmaceutiche
Università di Padova
Dr. Francesco Tisato
Dr.ssa Marina Porchia
Dr. Fiorenzo Refosco,
Dr.ssa Cristina Bolzati
ICIS-CNR, Padova
Prof. Rasika Dias
Department of Chemistry and Biochemistry
The University of Texas at Arlington (USA) 41